Innate immune cells constantly evaluate host mucosal surfaces and peripheral tissues for signs of infection or injury. The host must find a balance between tolerance of beneficial microorganisms and minor non-pathological microbial encounter vs. the development of a robust immune response to more serious infections. Emerging evidence suggests that this decision is made by the cell based on the strength and combination of signals it receives from its engagement with microorganisms and endogenous stimuli. These signals are sensed primarily by various classes of pattern recognition receptors (PRR), and while there has been remarkable progress in characterizing the individual signaling pathways induced by these receptors, relatively few studies have addressed how immune cells integrate combined PRR inputs and the combination of these signals with others arising from soluble host derived substances such as cytokines, lipids, and complement components. To evaluate the macrophage response to combined microbial stimuli, we have profiled the response of murine macrophages to single and pairwise combinations of toll-like receptor (TLR) ligands that induce either one or both of the MyD88 and TRIF branches of the TLR signaling pathway. We find predominantly less than additive levels of mRNA induction in response to ligands that both activate Myd88, likley due to saturation of shared signaling effectors in this pathway. On the other hand, in ligand combinations activating both Myd88 and TRIF, a select subset of key immune response mediators are induced to greater than additive levels. We predict that this synergistic response requires crosstalk between the adapter pathways, and that it signals to the host the presence of a more dangerous pathogenic challenge. We seek to determine the molecular mechanisms underlying this selective pattern of non-linear macrophage responses to combined ligands, as it would provide important insight to signaling pathway crosstalk during a microbial infection. This year, we completed a screen to identify regulators of dual TLR ligand-induced IL-6 secretion, an important link between the innate and adaptive immune systems. We previously identified approximately 200 genes with characteristics that could implicate them as regulators of the synergistic production of IL6 by dual TLR-activated macrophages. These genes were targeted in an siRNA screen, and approximately 30 putative hits were subjected to high throughput qPCR analysis to determine if they selectively affect different classes of TLR-induced inflammatory mediators. This has identified four novel genes that selectively regulate sustained TLR-induced transcription. All four genes contain DNA binding motifs suggesting that they likely function to modify the macrophage chromatin to permit sustained expression of immune effectors. These genes are not well characterized in the literature, and may provide novel therapeutic targets for regulating TLR-driven outputs. To further address how the TLR signaling network might mediate responses specific to combined TLR stimuli, we have investigated the localization dynamics of proximal pathway components in response to single vs. combined ligands. We observed unexpectedly that mouse IRAK1 forms clusters in the cytoplasm upon dual TLR ligand stimulation or with very high concentrations of single ligand. Interestingly, cells enriched in these IRAK1 clusters have low MAP kinase activity and lower activation of transcription factors like pATF2. IRAK1 clusters also colocalize with TLR signaling proteins distal in the pathway to IRAK1, suggesting that IRAK1 may function to sequester these components. Furthermore, in IRAK1 KO cells, phosphoprotein and cytokine responses to single TLR ligands are comparable to WT cells, but responses of IRAK1 KO cells to combined TLR ligands are increased. We conclude from these data that contrary to the traditional view of TLR signaling in mouse macrophages, IRAK1 may attenuate TLR responses in mouse macrophages under conditions of high pathogen load. In related work that arose from our RNAi screening of the TLR pathways (project AI001106), we determined that human and mouse macrophages exhibit different dependencies on the IRAK proteins in response to single TLR ligands (Sun et al (2016) Sci Signal. 9: ra3). Whereas single TLR ligand responses in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages strongly depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening could be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic manipulation of human TLR-dependent outputs. Further insight to the transcriptional programs activated by the Myd88 and TRIF-dependent signaling pathways has arisen this year from our study of hits identified in our genome-wide screens of the LPS response in human macrophages (project AI001106). Among the negative regulators of LPS-induced TNF transcription, we identified the interferon-induced gene IFIT1. Transcriptomic analysis of IFIT1 depleted cells suggested this protein is a reciprocal control switch between the Myd88-driven proinflammatory and the TRIF-driven interferon gene programs, as IFIT1 perturbation leads not only to increased inflammatory gene expression (such as TNF), but also to attenuated IFNb and interferon stimulated gene expression. We find a novel role for IFIT1 in promoting association of an HDAC2-SAP25 transcriptional regulation complex at LPS-induced gene promoters, which underlies the screen phenotype. We also find that attenuated IFNb production in IFIT1-depleted cells leads to increased susceptibility to bacterial infection. In further studies of the relative roles of NFkB and MAPK signaling induced by the Myd88 and TRIF pathways in the response to LPS, we have collaborated with LSB colleagues to determine how macrophages grade their transcriptional and inflammatory cytokine responses in the face of homeostatic and pathogenic levels of microbial stimuli. This work has identified distinct thresholds for NFkB and MAPK activation that provide sequential barriers for the production of inflammatory mediators to discriminate between varying levels of microbial danger signals (Gottschalk et al (2016) Cell Syst. 2: 378). To assess combined TLR activation in the context of intact bacterial infection, we have previously developed an infection model using the bacterium Burkholderia cenocepacia (Bcc), an opportunistic pathogen particularly problematic in immune compromised patients (Al-Khodor et al. Cell Microbiol. 16(3):378-95). This year we took advantage of the pathogenesis characteristics of Bcc to develop high content imaging assays that permit robust quantification of multiple stages of Bcc infection of macrophages (Miller et al. (2015) Assay Drug Dev Technol. 9: 515). Using this assay and imaging platform, we confirmed an important role for the AGS3 protein in regulating Bcc infection levels through effects on lysosome biogenesis (Vural et al. (2016) J Immunol. 196: 846), and we are also conducting RNAi screens in Bcc infected cells to assess the contribution of components from several PRR pathways (including TLRs) in the host macrophage response to intracellular bacterial infection.